Treatment experiences with focus on IL-6R inhibition in patients with VEXAS-syndrome and a case of remission with azacytidine treatment.

OBJECTIVES: The aim of the study was to evaluate the treatment response to Interleukin-6-receptor inhibitition (IL-6Ri), primarily tocilizumab, in patients with VEXAS. METHODS: Data were obtained from review of hospital based clinical records and included symptoms, laboratory data, transfusion history, pathology reports, imaging, and treatment. RESULTS: Fifteen patients were treated with tocilizumab intravenously. Two patients changed treatment to subcutaneous sarilumab. Three discontinued treatment due to treatment failure.Of the 10 patients with treatment-response and prednisone use prior to IL-6Ri one was tapered off prednisone, one used it intermittently, and seven patients could be reduced to 10 mg or less daily.Three patients exhibited a marked decrease in UBA1-levels during IL-6Ri which corresponded with symptom control and normalization of haemoglobin levels. However, in most a progressive marrow failure occurred as indicated by decreasing platelet levels, increasing MCV, and for some, declining haemoglobin levels and transfusion dependence in spite of control of the inflammatory symptoms and low c-reactive protein levels.One patient became refractory to both tocilizumab and sarilumab, and had previously failed conventional DMARDs, JAK-inhibition, TNFa-inhibition, and interleukin-1R-inhibiton. Treatment with 9 cycles of azacytidine resulted in complete symptom remission, discontinuation of prednisone, normalization of biochemical parameters and undetectable UBA1 mutation levels which has now lasted for 10 months since cessation of azacytidine. CONCLUSION: IL-6Ri induces control of inflammatory symptoms and allows decreased prednisone usage in a large subset of VEXAS patients. However, most experience progressive bone marrow failure during IL-6Ri.Azacytidine could be a promising treatment strategy and warrants further investigation.

Gender- and age-specific rates of heart failure and other adverse cardiovascular outcomes in systemic sclerosis.

OBJECTIVE: To investigate the long-term rates of heart failure (HF) and other adverse cardiovascular outcomes, including arrhythmias, myocardial infarction, ischaemic stroke, venous thromboembolism, pulmonary hypertension and pericarditis, in SSc patients according to gender and age. METHODS: Using Danish nationwide registries, SSc patients (diagnosed from 1996 to 2018) were matched with four controls from the background population by gender, age and comorbidities. Cox regression was used to compare the rates of cardiovascular outcomes between SSc patients and controls and the rate of mortality between SSc patients developing HF and HF patients without SSc, according to gender and age (above/below median). RESULTS: In total, 1569 SSc patients were matched with 6276 non-SSc controls (median age 55 years, 80.4% women, median follow-up 7.3 years). SSc had a higher rate of HF in both women [HR 2.99 (95% CI 2.18, 4.09)] and men [HR 3.01 (1.83, 4.95)] (Pinteraction = 0.88), with similar trends for other cardiovascular outcomes. SSc had a higher rate of HF in patients <55 years of age [HR 4.14 (95% CI 2.54, 6.74)] and ≥55 years [HR 2.74 (1.98, 3.78)] (Pinteraction = 0.22), with similar trends for other cardiovascular outcomes. SSc patients with new-onset HF had a higher rate of mortality than HF patients without a history of SSc, irrespective of gender (Pinteraction = 0.53) and age (Pinteraction = 0.43). CONCLUSIONS: SSc was associated with higher rates of HF and other cardiovascular outcomes than matched controls, irrespective of gender and age. Among patients with new-onset HF, a history of SSc was associated with higher mortality.

Human herpesvirus infections and circulating microvesicles expressing galectin-3 binding protein in patients with systemic lupus erythematosus.

OBJECTIVES: Circulating microvesicles (MVs) expressing the type 1 interferon-inducible protein galectin-3 binding protein (G3BP) are potentially major sources of autoantigens in systemic lupus erythematosus (SLE). In this study, we explore if plasma concentrations of G3BP-expressing MVs correlate with signs of various active human herpesvirus (HHV) infections in SLE patients, suggesting a virus-induced mechanism for the generation of these vesicles. METHODS: In 49 SLE patients, the plasma levels of immunoglobulin G (IgG) against cytomegalovirus (CMV) pp52, Epstein-Barr virus (EBV) early antigen diffuse (EA/D), and HHV6 p41 were measured by ELISAs and used as humoral markers of ongoing/recently active viral infection. MVs in platelet-poor plasma were quantified and characterised by flow cytometry, with regard to the binding of Annexin V (AnxV) and the expression of G3BP. Spearman's rho and the Wilcoxon rank-sum test were applied for associative evaluation of virus serology with MV subsets, and clinical and demographic data. RESULTS: The CMV pp52-directed antibodies correlated positively with the high G3BP-expressing MVs; either low (rho=0.4, p-value=0.005) or high (rho=0.37, p-value=0.01) in AnxV-expression. Furthermore, these MV subsets were higher in individuals with high and low IgG levels against CMV pp52 and EBV EA/D, respectively, relative to subjects with low and high IgG levels against these HHV antigens. Importantly, none of the associations were explained by immunosuppressants or antimalarials. CONCLUSIONS: Ongoing/recently active CMV infection is associated with circulating MVs expressing G3BP in SLE patients, supporting a link between specific viral infections and potentially pathogenic MVs in SLE.

Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Decreased platelet size is associated with platelet activation and anti-phospholipid syndrome in systemic lupus erythematosus.

Objectives: . SLE is an autoimmune disease with increased cardiovascular morbidity and platelet activation. In the general population, increased platelet size predicts platelet reactivity and cardiovascular disease. The aim of this study was to investigate whether platelet size related to platelet activation and cardiovascular disease in SLE. Methods: . Fresh blood samples from SLE patients ( n = 148), healthy volunteers ( n = 79) and disease controls ( n = 40) were analysed for platelet size and activation by flow cytometry, ELISA and cell count. Associations to manifest cardiovascular disease, venous thrombosis and APS were adjusted for traditional cardiovascular risk factors using logistic regression analysis. Results: . SLE patients had decreased platelet size as compared with healthy controls ( P = 0.003). In SLE, decreased platelet size was related to increased platelet activation, in particular microparticle formation ( P < 0.0001, r = -0.46) and release of serotonin from dense granules ( P < 0.001, r = 0.57). SLE patients with aCL had decreased platelet size ( P = 0.02) and aCL decreased platelet size in vitro ( P = 0.007). In contrast to the general population, increased platelet size was not associated with cardiovascular disease. Instead, decreased platelet size was associated with secondary APS, even after adjusting for traditional cardiovascular risk factors ( P = 0.01, odds ratio 3.58). Conclusion: . Platelet size is decreased in SLE patients and associated with microparticle formation and APS. Future studies are needed to determine the underlying mechanism(s) as well as the potential predictive value of small platelets for disease complications in SLE.

A review of studies of the proteomes of circulating microparticles: key roles for galectin-3-binding protein-expressing microparticles in vascular diseases and systemic lupus erythematosus.

Subcellular microvesicles (MVs) have attracted increasing interest during the past decades. While initially considered as inert cellular debris, several important roles for MVs in physiological homeostasis, cancer, cardiovascular, and autoimmune diseases have been uncovered. Although still poorly understood, MVs are involved in trafficking of information from cell-to-cell, and are implicated in the regulation of immunity, thrombosis, and coagulation. Different subtypes of extracellular MVs exist. This review focuses on the cell membrane-derived shedded MVs (ranging in size from 200 to 1000 nm) typically termed microparticles (MPs). The numbers and particularly the composition of MPs appear to reflect the state of their parental cells and MPs may therefore carry great potential as clinical biomarkers which can be elucidated and developed by proteomics in particular. Determination of the identity of the specific proteins and their quantities, i.e. the proteome, in complex samples such as MPs enables an in-depth characterization of the phenotypical changes of the MPs during disease states. At present, only a limited number of proteomic studies of circulating MPs have been carried out in healthy individuals and disease populations. Interestingly, these studies indicate that a small set of MP-proteins, in particular, overexpression of galectin-3-binding protein (G3BP) distinguish MPs in patients with venous thromboembolism and the systemic autoimmune disease, systemic lupus erythematosus (SLE). G3BP is important in cell-cell adhesion, clearance, and intercellular signaling. MPs overexpressing G3BP may thus be involved in thrombosis and hemostasis, vascular inflammation, and autoimmunity, further favoring G3BP as a marker of "pathogenic" MPs. MPs expressing G3BP may also hold a potential as biomarkers in other conditions such as cancer and chronic viral infections. This review highlights the methodology and results of the proteome studies behind these discoveries and places them in a pathophysiological and biomarker perspective.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Cross-sectional study of soluble selectins, fractions of circulating microparticles and their relationship to lung and skin involvement in systemic sclerosis.

BACKGROUND: Endothelial damage and activation may play central roles in the pathogenesis of systemic sclerosis (SSc) and are reflected by microparticles (MPs) and soluble selectins. The objective of this study was to determine if these potential biomarkers are associated with specific organ involvements or cutaneous subgroups of SSc patients. METHOD: MPs in platelet-poor plasma from 121 patients with SSc, 79 and 42 with limited and diffuse cutaneous disease, respectively, were characterized by flow cytometry for their capacity to bind annexin V in combination with surface markers of either platelets (PMPs), leukocytes (LMPs) or endothelial cells (EMPs). Soluble E- and P-selectin levels were determined in plasma. By correlation analyses, this was held against involvement of skin, lung function, lung fibrosis, pulmonary artery hypertension, and serology. RESULTS: None of the markers were associated with cutaneous subgroups of SSc. Concentrations of annexin V non-binding EMPs and annexin V non-binding LMPs were negatively correlated to pulmonary diffusing capacity (DLCO) (r = -0.28; p = 0.003; r = -0.26; p = 0.005) and forced vital capacity (FVC) (r = -0.24; p = 0.009; r = -0.29; p = 0.002), driven by patients with limited and diffuse cutaneous disease, respectively. Soluble E-selectin levels correlated negatively to DL(CO) (r = -0.21, p = 0.03) and FVC (r = -0.25; p = 0.007); and soluble P-selectin correlated negatively to DL(CO) (r = -0.23, p = 0.01). CONCLUSION: Negative correlations between annexin V non-binding EMP and LMP concentrations with lung function parameters (DL(CO) and FVC) differed between limited and diffuse cutaneous subsets of SSc, indicative of various pathogeneses of lung involvement in SSc, possibly with a differential role of MPs.

Unique protein signature of circulating microparticles in systemic lupus erythematosus.

OBJECTIVE: To characterize the unique qualities of proteins associated with circulating subcellular material in systemic lupus erythematosus (SLE) patients compared with healthy controls and patients with other chronic autoimmune diseases. METHODS: Using differential centrifugation and high-sensitivity nano-liquid chromatography tandem mass spectrometry, we systematically profiled proteins of microparticles (MPs) from SLE patients (n=12), systemic sclerosis (SSc) patients (n=6), and rheumatoid arthritis (RA) patients (n=6), as well as healthy controls (n=12). RESULTS: We identified 531 unique proteins and showed that the differences between healthy controls and patients with SLE with regard to the abundance of 248 proteins were highly statistically significant. Almost half of the proteins that were increased by >2-fold were complement proteins and Ig (increased by 100-4,000 times). MP Ig and complement loads also distinguished SLE from RA and SSc and correlated strongly with clinical SLE severity. Subsets of microtubule proteins, fibronectin, 14-3-3η, and desmosomal proteins as well as ficolin 2 and galectin 3 binding protein were also highly increased. In SLE MPs, levels of cytoskeletal, mitochondrial, and organelle proteins, including lysosome-associated membrane protein 1 and transforming growth factor ß1, were decreased. CONCLUSION: The data show that SLE patients have increased numbers of MPs that are heavily tagged for removal and fewer MPs with normal protein composition. SLE MPs are unique and specific proteins that represent novel leads for our understanding of SLE and for the development of new treatments of the disease.

Circulating microRNA expression profiles associated with systemic lupus erythematosus.

OBJECTIVE: To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined using quantitative reverse transcription-polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients. RESULTS: Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142-3p (miR-142-3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342-3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10(-9) ). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis. CONCLUSION: Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor ß signaling pathways. Other targets include regulation of apoptosis, cytokine-cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.

Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation.

OBJECTIVE: To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters. METHODS: Sixty-eight clinically well-characterized SLE patients, 38 healthy controls, 6 patients with systemic sclerosis (SSc), and 6 patients with rheumatoid arthritis (RA) were included. The numbers of annexin V-binding MPs displaying IgG, IgM, or C1q were enumerated by flow cytometry. MP protein levels were determined by mass spectrometry in clinically defined subsets of SLE patients and controls. The MP IgG load was determined by flow cytometric analysis of all samples from SLE patients and healthy controls. RESULTS: SLE patients had significantly increased total and relative numbers of IgG-positive MPs (P = 0.0004), with a much higher average IgG load per MP (P < 0.0001) than healthy controls. Quantitative mass spectrometry of purified MPs verified significantly increased IgG, IgM, and C1q levels in SLE patients. In RA and SSc patients, the average IgG load per MP was significantly lower than in SLE patients (P = 0.006 and P = 0.05, respectively). Also, the IgM load and C1q load per MP were significantly higher in SLE patients than in the control groups (P < 0.05), except for IgM in the RA group. IgG-positive MPs were significantly associated with the presence of anti-double-stranded DNA, anti-extractable nuclear antigen, and antihistone antibodies, with total IgG, and with decreased leukocyte counts. Average IgG load per MP was associated with lower concentrations of MPs, the presence of anti-C1q antibodies, and complement consumption. CONCLUSION: Our findings indicate that circulating cell-derived MPs in SLE patients carry increased loads of IgG, IgM, and C1q and that IgG MPs are associated with autoantibodies and complement activation. The findings link immunologic reactions on MPs with the etiology of SLE.

Quantitative proteome profiling of normal human circulating microparticles.

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.

Distinct features of circulating microparticles and their relationship to clinical manifestations in systemic lupus erythematosus.

OBJECTIVE: Characterization of the abundance, origin, and annexin V (AnxV)-binding capabilities of circulating microparticles (MPs) in SLE patients and healthy controls and to determine any associations with clinical parameters. METHODS: Seventy unselected SLE patients and 29 sex- and age-matched healthy control subjects were included in the study. MPs were isolated from citrate-treated plasma and characterized by flow cytometry using AnxV or antibodies to platelet, leukocyte, or endothelial cell surface markers. RESULTS: SLE patients had significantly increased concentrations of AnxV-nonbinding (AnxV-) MPs (P<0.0001), while the concentrations of total MPs (P=0.011) and AnxV-binding (AnxV+) MPs (P<0.0001) were decreased, as compared with controls. Based on flow cytometric characteristics, 2 subgroups of AnxV- MPs could be discerned: AnxV- cell-derived MPs (CDMPs) and AnxV- MPs of unknown nature (UNMPs). Both fractions were significantly increased in SLE patients (P=0.007 and P=0.0018, respectively). Platelet- and leukocyte-derived MPs were decreased in the SLE patients (P<0.0001), whereas no difference was observed for endothelial cell-derived MPs (P=0.14). The concentrations of AnxV- CDMPs correlated with the concentrations of endothelial cell-derived MPs, the disease activity score, active nephritis, hypertension, history of arterial thrombosis, and triglyceride levels (P<0.05 for all comparisons). CONCLUSION: The concentrations and composition of MPs in SLE patients differ markedly from those in healthy subjects. Overall MP numbers were significantly decreased, but two distinct subpopulations of AnxV- MPs were significantly increased. These findings call for further characterization of MPs in SLE patients to elucidate their role in disease pathogenesis.

Role of Neutrophil Extracellular Traps Regarding Patients at Risk of Increased Disease Activity and Cardiovascular Comorbidity in Systemic Lupus Erythematosus.

OBJECTIVE: Neutrophil extracellular traps (NET) are essential in host defense, but are also linked to inflammation and autoimmunity, including in systemic lupus erythematosus (SLE). We recently described that immune complexes (IC) induce NET formation, promoting SLE-like disease in mice. In the current study, we investigated, for the first time to our knowledge, the role of NET in human SLE and their association with disease activity and severity. METHODS: Levels of NET (myeloperoxidase-DNA complexes) were analyzed in plasma from 4 cross-sectional SLE cohorts (n = 44-142), 1 longitudinal SLE cohort (n = 47), and healthy individuals (n = 100) using ELISA. Type I interferon activity was determined using a cell reporter system. RESULTS: Patients with SLE had elevated levels of NET in circulation compared to healthy controls (p < 0.01). NET levels identified patients with a severe disease phenotype characterized by IC-driven nephritis (p < 0.05). Though not associated with current disease activity (p = 0.20), levels of NET were associated with future increase in the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) within 3 months (OR 1.75, p = 0.01), as well as an overall heightened SLEDAI over 1 year (p < 0.01). Finally, levels of NET were associated with arterial events (OR 5.0, p = 0.02) and endothelial cell activation (p < 0.001). CONCLUSION: NET levels are elevated in patients with SLE, associated with IC-driven disease. NET levels provide significant clinical value in identifying patients at risk of active disease and/or severe disease, including nephritis and cardiovascular disease, and may allow for early interventions.

Autoantibodies to Cytosolic 5'-Nucleotidase 1A in Primary Sjögren's Syndrome and Systemic Lupus Erythematosus.

Introduction: Autoantibodies to cytosolic 5'-nucleotidase 1A (cN-1A; NT5C1A) have a high specificity when differentiating sporadic inclusion body myositis from polymyositis and dermatomyositis. In primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE) anti-cN-1A autoantibodies can be detected as well. However, various frequencies of anti-cN-1A reactivity have been reported in SLE and pSS, which may at least in part be explained by the different assays used. Here, we determined the occurrence of anti-cN-1A reactivity in a large number of patients with pSS and SLE using one standardized ELISA. Methods: Sera from pSS (n = 193) and SLE patients (n = 252) were collected in five European centers. Anti-cN-1A, anti-Ro52, anti-nucleosome, and anti-dsDNA reactivities were tested by ELISA (Euroimmun AG) in a single laboratory. Correlations of anti-cN-1A reactivity with demographic data and clinical data (duration of disease at the moment of serum sampling, autoimmune comorbidity and presence of muscular symptoms) were analyzed using SPSS software. Results: Anti-cN-1A autoantibodies were found on average in 12% of pSS patients, with varying frequencies among the different cohorts (range: 7-19%). In SLE patients, the anti-cN-1A positivity on average was 10% (range: 6-21%). No relationship was found between anti-cN-1A reactivity and the presence or absence of anti-Ro52, anti-nucleosome, and anti-dsDNA reactivity in both pSS and SLE. No relationship between anti-cN-1A reactivity and duration of disease at the moment of serum sampling and the duration of serum storage was observed. The frequency of muscular symptoms or viral infections did not differ between anti-cN-1A-positive and -negative patients. In both disease groups anti-cN-1A-positive patients suffered more often from other autoimmune diseases than the anti-cN-1A-negative patients (15 versus 5% (p = 0.05) in pSS and 50 versus 30% (p = 0.02) in SLE). Conclusion: Our results confirm the relatively frequent occurrence of anti-cN-1A in pSS and SLE patients and the variation in anti-cN-1A reactivity between independent groups of these patients. The explanation for this variation remains elusive. The correlation between anti-cN-1A reactivity and polyautoimmunity should be evaluated in future studies. We conclude that anti-cN-1A should be classified as a myositis-associated-, not as a myositis-specific-autoantibody based on its frequent presence in SLE and pSS.

Efficient evaluation of humoral immune responses by the use of serum pools.

BACKGROUND: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. METHODS: We created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögren's syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. RESULTS: The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. CONCLUSION: We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors.

"Kill" the messenger: Targeting of cell-derived microparticles in lupus nephritis.

Immune complex (IC) deposition in the glomerular basement membrane (GBM) is a key early pathogenic event in lupus nephritis (LN). The clarification of the mechanisms behind IC deposition will enable targeted therapy in the future. Circulating cell-derived microparticles (MPs) have been proposed as major sources of extracellular autoantigens and ICs and triggers of autoimmunity in LN. The overabundance of galectin-3-binding protein (G3BP) along with immunoglobulins and a few other proteins specifically distinguish circulating MPs in patients with systemic lupus erythematosus (SLE), and this is most pronounced in patients with active LN. G3BP co-localizes with deposited ICs in renal biopsies from LN patients supporting a significant presence of MPs in the IC deposits. G3BP binds strongly to glomerular basement membrane proteins and integrins. Accordingly, MP surface proteins, especially G3BP, may be essential for the deposition of ICs in kidneys and thus for the ensuing formation of MP-derived electron dense structures in the GBM, and immune activation in LN. This review focuses on the notion of targeting surface molecules on MPs as an entirely novel treatment strategy in LN. By targeting MPs, a double hit may be achieved by attenuating both the autoantigenic fueling of immune complexes and the triggering of the adaptive immune system. Thereby, early pathogenic events may be blocked in contrast to current treatment strategies that primarily target and modulate later events in the cellular and humoral immune response.

Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies.

OBJECTIVE: In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore, FLCs' association with Epstein-Barr virus (EBV) antibodies was examined. METHODS: Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations of serum FLCs due to decreased clearance. RESULTS: Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001) also after adjusting for Ig levels (p<0.0001). The concentration of serum FLCs correlated with a global disease activity (SLE disease activity index (SLEDAI)) score of the SLE patients (r = 0.399, p = 0.007). Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009), and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total immunoglobulin (IgG and IgA) concentrations correlated with FLC concentrations and elevated FLC levels were additionally shown to associate with the inflammatory marker C-reactive protein and also with complement consumption determined by low C4 in SLE patients. Collectively, results indicated that elevated serum FLCs reflects increased B cell activity in relation to inflammation. SLE patients had an increased seropositivity of EBV-directed antibodies that did not associate with elevated FLC concentrations. An explanation for this could be that serum FLC concentrations reflect the current EBV activity (reactivation) whereas EBV-directed antibodies reflect the extent of previous infection/reactivations. CONCLUSION: SLE patients have elevated concentrations of serum FLCs that correlate with global disease activity scores and especially serologic markers for active disease. These findings are suggestive of circulating FLCs having potential as a new supplementary serologic biomarker in SLE.

Ficolins and the lectin pathway of complement in patients with systemic lupus erythematosus.

The complement system plays a pathophysiological role in systemic lupus erythematosus (SLE). This study aims to investigate whether an association exists between the ficolins that are part of the lectin complement pathway and SLE. EDTA plasma samples from 68 Danish SLE patients and 29 healthy donors were included in the study. Plasma concentrations of Ficolin-1, -2, and -3 were determined in specific sandwich ELISAs. Lectin pathway activity via Ficolin-3 was measured in ELISA on acetylated bovine serum albumin (acBSA) and measured as Ficolin-3 binding and deposition of C4, C3 and the terminal complement complex (TCC). SLE patients had increased levels of Ficolin-3, 21.6µg/ml as compared to 17.0µg/ml in healthy controls (P=0.0098). The Ficolin-1 plasma concentration was negatively correlated with SLE Disease Activity Index (SLEDAI) (Rho=-0.29, P=0.015) and positively correlated to the [Systemic Lupus International Collaborating Clinics (SLICC)/American College of Rheumatology (ACR) Damage Index] (SDI) (Rho=0.27, P=0.026). The Ficolin-1 concentration was also associated with the occurrence of arterial (P=0.0053) but not venous thrombosis (P=0.42). Finally, deposition of C4, C3 and TCC in the Ficolin-3 pathway were all correlated to SLEDAI, respectively (P<0.0076). The Ficolin-1 association to SLEDAI and SDI as well as arterial thrombosis shown in this study suggests that Ficolin-1 may be a potential new biomarker for patients with SLE. Furthermore, Ficolin-3 mediated complement activation may be valuable in monitoring disease activity in SLE patients due to the high sensitivity for complement consumption in the assay independent of the Ficolin-3 concentration.